The identification of functional receptors /ion channels in bladder urothelial (UT) cells and the involvement, in turn, of these in the release of chemical mediators (acetylcholine, ATP, substance P) suggest that UT cells exhibit specialised sensory and signalling properties. In addition, stimulation of various 'sensor molecules' (i.e. muscarinic, purinergic, neurokinin and TRP channels, stretch receptors) can all elicit release of a number of neurotransmitters from UT cells. However, there is virtually no information as to the mechanism(s) for this release. The goals of this proposal are to utilize fluorescent FM dyes to track the movement of stimulus evoked dye -filled vesicles in order to provide information as to the trafficking events underlying transmitter release by UT cells. While FM dyes have been utilized in neurons as an 'activity marker', their use in UT cells to correlate vesicle traffic and release mechanisms, is novel and should provide new information as to how UT cells respond to chemical and mechanical stimuli. Specific Aim I will characterize chemical versus mechanical evoked vesicle trafficking in urothelial cells. Specific Aim II will evaluate the intracellular mechanisms underlying urothelial cell vesicular trafficking responses to chemical and mechanical stimuli, the cytosolic location of vesicular pools and the identity (to a limited degree) of the vesicular cargo. Specific Aim III will evaluate the vesicle trafficking profile in urothelial following pathology. We and others have shown that injury or inflammation results in augmented release of chemical- or stretch-evoked neurotransmitters from the urothelium. Alterations in urothelial-derived mediators could alter the activity of bladder nerves in close proximity to the UT and impact bladder function. Thus, in this aim, we will examine how inflammation alters stimulus-evoked vesicular traffic. Results from these studies will provide information as to how urothelial cells receive and integrate multiple stimuli.